Summary: (1) Goals of Project: - To identify a carrier which will increase the immunogenicity of the HIV-1 subunits, and will be able to recall anti-HIV B memory cells in patients with pre- existing immune deficiency. - To identify a carrier which would augment the TH1/TH2 ratio of infected individuals and will favor generation of cellular responses including cytotxic cells (CTL) and beta chemokines production. (2) Experimental approaches: - The gram negative Brucella abortus (Ba), and LPS derived from its cell wall (Ba- LPS), were tested as carriers for either inactivated HIV-1 virions, gp120 (SF2) glycoprotein, or peptide derived from the V3-loop of HIV-1 (MN) env. The different conjugates were used to immunize mice with different degrees of T cell deficiency. Four Rhesus macaques were also vaccinated with a Ba-V3 conjugate. - Both humoral and cytotxic immune responses were measured including sytemic and mucosal antibody responses. Biologically relevant antibodies were measured in syncytia inhibition assays. - In vitro studies with human T cells and elutriated monocytes from normal as well as HIV-1 infected patients were assessed for their lymphokine production in response to Ba and Ba-LPS by PCR and biological assays. - PCR primers were designed to test the ability of Ba ,Ba-LPS,and Ba/DNA, to elicit chemokines from human PBL (3) Major findings: - Ba conjugated to a peptide containing B-cell epitope and CTL epitope (N3v3), generated both neutralizing antibodies and cytotoxic T cells capable of killing HIV-infected targets. CD4 - depleted mice retained their ability to generate anti-HIV neutralizing Ab and CTL after immunization with the Ba-N3V3 conjugate. - Ba and Ba-LPS were found to directly activate purified human CD4-positive TH1 cells, and to a lesser degree, CD8-positive cells, as judged by induction of the lymphokines IL2 and IFN-gamma. PBL from HIV-1 infected individuals were also responsive. Ba and Ba-LPS can also activate IL12 secretion by human monocytes. - Rhesus macacques produced high titer HIV-1-neutralizing antibodies in the serum and mucosal surfaces.In addition,IFNgama producing cells and beta-chemokine producing cells were measured after Ba-gp120 immunization. - Inactivated Ba , as well as Ba derived DNA and LPS were found to induce mRNA for RANTES, MIP-1a, and MIP-1b in human PBL (in 20 hr) , and secretion of these chemokines from CD8+ and CD4+ cells and from monocytes. These chemokines will add to the anti-viral millieu in vaccinated individuals. The beta chemokines can block and prevent infection of R5 viruses by binding and/or downregulation of the CCR5 HIV-1 coreceptor. - new efforts are underway to clone HIV-1 sructural genes (Gag/Pol) into plasmid and infect attenuated strain of Ba, used as a veterenary vaccine. The bacterium containing the recombinant plasmid will be killed by heat and will be tested for generation of CTL in BALB/c mice and rhesus macaques.